1. Field of the Invention
The present invention relates to the detection of biological substances through binding interactions and, in particular, to methods of detecting proteins and other substances by mediated catalytic electrochemistry.
2. Description of the Related Art
For many reasons, researchers are interested in the detection of biological substances such as nucleic acids, proteins, and carbohydrates. Detection of such biomolecules can allow for identification and development of targets for drug discovery and gene expression analysis. The electrochemical detection of nucleic acids provides an alternative to fluorescent and radiochemical detection techniques that potentially eliminates the need for labeling.
The parent applications of the instant application, whose entire specifications, drawings, and claims are specifically incorporated herein by reference, disclose, among other inventions, sequencing and methods of qualitatively and quantitatively detecting nucleic acid hybridization. Such inventions represent a major advance in the art and provide oxidation-reduction reactions that function in a catalytic manner without the addition of an enzyme or fluorescent label. These catalytic reactions are useful for determining the presence or absence of nucleic acids and provide for extremely accurate testing of biological samples. More specifically, catalytic oxidation has been found to be useful for quantitative detection of preselected nucleic acid bases (U.S. Pat. No. 5,871,918). The disclosures of each of the patents and publications referred to herein are incorporated herein by reference.
The technology described in U.S. Pat. No. 5,871,918 utilizes the discovery that nucleotide bases of DNA can be electrochemically oxidized using transition metal complexes as mediators. In this system, the nucleotide bases function as an array of endogenous redox-active labels that allow for ultrasensitive detection of DNA in conjunction with microelectrode methods. The detection reaction follows a two-step mechanism involving reversible oxidation/reduction of the mediator. First the mediator is oxidized by an electrode. Then, the mediator is reduced by the preselected nucleotide base and reoxidized at the electrode. In order for mediated oxidation of nucleic acids to proceed efficiently, the mediator and nucleotide base should have similar oxidation potentials. For example, catalytic oxidation of guanine can be carried out using the mediator, ruthenium2+ (2,2xe2x80x2-bipyridine)3 (Ru(bpy)32+ ). In solution, Ru(bpy)32+ exhibits a reversible redox couple at 1.05 V (vs. Ag/AgCl reference), similar to the oxidation potential observed for guanine (about 1.1 V vs. Ag/AgCl). Thus, addition of guanine-containing DNA to a solution of Ru(bpy)32+ leads to catalytic enhancement in the electrochemical oxidation current via the following reaction sequence:
Ru(bpy)32+xe2x86x92Ru(bpy)33++e-
Ru(bpy)33++DNA xe2x86x92DNAox+Ru(bpy)32+
where DNAox represents a DNA molecule in which guanine has undergone a one electron oxidation.
The regeneration of reduced Ru(bpy)32+ by reaction with guanine creates a catalytic cycle in which the presence of DNA is detected by transfer of electrons from the preselected base to the electrode. The number of turnovers obtained in the catalytic cycle depends on the number of electrons in the preselected base that can be oxidized by the mediator and the number of preselected bases. In the case of guanine oxidation, Ru(bpy)32+ is capable of oxidizing guanine by at least two electrons (Armistead, P. M. and Thorp, H. H., Anal. Chem. 2000, 72, 3764), and some reports suggest as many as 30 electrons obtained from guanine through overoxidation steps (Thorp, H. H., Trends Biotechnol. 1998, 16, 117). A typical DNA molecule will contain on average about one guanine every four bases so even a small oligonucleotide will have multiple guanines available for catalytic turnover of Ru(bpy)32+. As a result of these properties, detection of nucleic acids via mediated catalytic electrochemistry is an extremely sensitive method.
Thus, in one embodiment of the prior invention, a nucleic acid sample is contacted with an oligonucleotide probe, which possesses a sequence, at least a portion of which is capable of binding to a known portion of the sequence in the nucleic acid sample, to form a hybridized nucleic acid, after which the hybridized nucleic acid is reacted with a suitable mediator, which is capable of oxidizing a preselected nucleic acid base in the hybridized nucleic acid sample in an oxidation-reduction reaction.
The selection of mediator in this prior work is dependent upon the particular preselected nucleotide base chosen, and is readily determinable by those skilled in the art. Particularly preferred mediators include transition metal complexes that are capable of participating in electron transfer with the preselected base such that the reduced form of the metal complex is regenerated, completing a catalytic cycle. An example of a suitable transition metal complex is Ru(bpy)32+; however, the mediator or oxidizing agent may be any molecule such as a cationic, anionic, non-ionic, or zwitterionic molecule that is reactive with the preselected base at a unique oxidation potential to transfer electrons from the nucleic acid to the electrode. All that is required is that the mediator be reacted with the hybridized nucleic acid sample under conditions sufficient to achieve the selective oxidation of the preselected base.
The oxidation-reduction rate is detected, for example, with a detection electrode, and the electronic signal may be detected by cyclic voltammetry or other means known in the art. Hybridized DNA target contains guanine and is therefore more redox-active than the probe strand, which preferably is either selected or designed to contain a minimal number of guanines.
In U.S. Pat. No. 5,968,745 of Thorp et al., a polymer-electrode is provided that is useful for the electrochemical detection of a preselected base in a nucleic acid. The polymer-electrode comprises: (a) a substrate having a conductive working surface; and (b) a polymer layer on the conductive working surface. The polymer layer has a plurality of microfluidic reaction openings distributed throughout the layer. An oligonucleotide probe is preferably bound to the polymer layer.
U.S. Pat. No. 6,127,127 provides a self-assembled phosphonate monolayer, which in the preferred embodiment is a carboxy-alkyl phosphonate, on an ITO surface. The oligonucleotide probe is immobilized on an electrode surface modified by the self-assembled monolayer. The electrode with the self-assembled monolayer is useful for the electrochemical detection of a preselected base in a nucleic acid and for determining the presence of a target nucleic acid in a sample, by contacting the self-assembled monolayer with the sample, so that the target nucleic acid and the oligonucleotide probe form a hybridized nucleic acid on the monolayer; reacting the hybridized nucleic acid with a transition metal complex capable of oxidizing a preselected base in the hybridized nucleic acid in an oxidation-reduction reaction; detecting the oxidation-reduction reaction; and determining the presence or absence of the target nucleic acid from the detected oxidation-reduction reaction.
In both the polymer-electrode and monolayer patents, determination of the presence of a target protein in a sample can also be achieved and comprises attaching a protein-binding substance to a polymer-electrode or self-assembled monolayer on a conductive working surface according to the invention; exposing the polymer-electrode or monolayer to the sample; exposing the polymer-electrode or monolayer to a second protein-binding substance that has been modified to contain an oligonucleotide label; reacting the polymer-electrode or monolayer with a transition metal complex capable of oxidizing a preselected base in the oligonucleotide label in an oxidation-reduction reaction; detecting the oxidation-reduction reaction; and determining the presence or absence of the target protein from the detected oxidation-reduction reaction. The polymer-electrode or monolayer may be brought into contact with the conductive working surface of the substrate either before or after reacting the polmer-electrode or monolayer with the first protein binding substance. The target protein may be modified to contain an oligonucleotide label as is known in the art.
Amino acids, such as tyrosine and tryptophan, have been detected by direct, unmediated electrochemistry (Brabec, V. and Mornstein, V., Biochimica et Biophysica Acta, 1980, 625, 43; Renaud, J. A. et al., Bioelectrochem. and Bioenergetics, 1980, 7, 395). However, the levels of current obtained by direct, unmediated oxidation of amino acids are generally low, on the order of a few microamps for concentrated solutions of amino acids (100 xcexcM).
Oxidation potentials of several amino acids have been determined using thermodynamic and kinetic methods (DeFilippis, M. R. et al., Biochem., 1989, 28, 4857). The oxidation potential for tyrosine is about 0.6-0.73 V (vs. Ag/AgCl reference), and the oxidation potential for tryptophan is about 0.6-0.85 V (vs. Ag/AgCl). Other amino acid oxidation potentials have been estimated for histidine (1.1-1.4 V), cysteine (0.5-0.8 V), methionine (0.9-1.2 V), and cystine (1.1-1.2 V)(Brabec, V. and Mornstein, V., Biochimica et Biophysica Acta, 1980, 625, 43; Renaud, J. A. et al., Bioolectroehem. and Bioenergetics, 1980, 7, 395), but the extent to which these amino acids are oxidized depends on the electrode material.
It has been observed that proteins can be adsorbed to electrodes at both negative and positive potentials through an electrostatic interaction when the protein net charge is opposite that of the electrode (Brabec, V. et al., Bioelectrochem. and Bioenergetics, 1981, 8, 451). Although the adsorption is initiated by an electrostatic interaction, it has been found that adsorption in this manner leads to the formation of a protein that is irreversibly adsorbed to the electrode surface. This phenomenon can interfere with electrochemical detection at electrodes because the adsorbed protein blocks the electrode surface. Thus, in attempts to detect protein in solution directly, Brabec et al. found that the adsorbed protein fouled the electrode surface so that fresh protein molecules could not reach the surface and interfered with protein detection at the electrode. Along these same lines, Elbicki et al. (Elbicki, J. et al., Biosensors, 1989, 4, 251) have found that the removal of proteins from samples is necessary to protect electrode surfaces from fouling.
Because of the low sensitivity and poor selectivity of direct electrochemical detection of proteins, attempts have been made to use exogenous labels to facilitate this electrochemical detection. In one example, DiGleria et al. sought to convert a xe2x80x9credox-inactivexe2x80x9d protein to a xe2x80x9credox-activexe2x80x9d protein by adding an exogenous redox-active label to the enzyme xcex2-lactamase (DiGleria, K. et al., FEBS Letters, 1997, 400, 155). This approach involved engineering the enzyme to contain an unnatural cysteine residue and then modifying this residue with a thiol-reactive ferrocene compound, N-(2-ferrocene-ethyl)maleimide.
Previous work with metal complexes and amino acids includes the study of one-electron oxidation of tryptophan by ruthenium-DNA intercalator compounds (Wagenknecht, H.-A. et al., J. Amer. Chem. Soc., 2000, 122, 1). This process is not catalytic, however, and was initiated by light rather than by an applied electrochemical potential. Also, in this system, electron transfer between tryptophan and the ruthenium complex was found to be dependent on guanine as an intermediate. In another study, the one-electron oxidation of a reference redox couple, osmium2+ (2,2xe2x80x2-bipyridine)3 (Os(bpy)3), was used to determine the redox potentials of tryptophan and tyrosine by pulse radiolysis (DeFilippis, M. R. et al. Biochem., 1989, 28, 4857), but in this case, Os(bpy)32+ acted as a reductant of an oxidized amino acid. Therefore, a two-step catalytic oxidation reaction between oxidized transition metal complex and the amino acid was not present in this prior work.
Recently, Pikulski and Gorski have suggested the catalytic oxidation of disulfide bonds and the amino acid cystine by the iridium complex, Ir(H2O)2Cl2 (Pikulski, M. et al., Anal. Chem., 2000, 72, 2696). This chemistry has been utilized to create a flow injection sensor for detecting insulin, in which the mediator is immobilized in an oxide layer on a glassy carbon electrode and the insulin is in solution. A related method has been proposed for detecting amino acids and peptides in solution at copper electrodes using catalytic electrochemistry (Brazill, S. A. et al., Anal. Chem., 2000, 72, 5542). Although not fully understood, the detection process is believed to involve amino acid oxidation by Cu(III) in a conductive oxide or hydroxide layer that is formed under alkaline conditions. In both the work of Pikulski et al. and Brazill et al., the metal complex is a solid phase mediator in the electrode to which the analyte diffuses nonspecifically. Thus, the electrode is only capable of detecting analytes that come into direct contact with the electrode. The electrochemical detection utilized in the above methods is distinct from the mediated electrochemical detection methods of the instant invention wherein there is a nonconductive layer and wherein the mediator is a soluble, freely diffusible transition metal complex that is capable of oxidizing labels on binders that are bound to the electrode via biological binding interactions (antibody-protein, receptor-ligand, DNA-protein, and protein-protein).
A means of detecting protein binding has been disclosed in the PCT application of Fowlkes and Thorp using a soluble transition metal complex mediator and biomolecules labeled with transition metal complexes including Ru(bpy)32+ and Os(bpy)32+ (PCT/US98/02440). The basic mechanism of this detection scheme involves electron transfer from the label to an electrode via the soluble mediator. The electrochemical current enhancement obtained from the label is limited by the number of electrons in the label that can be oxidized by the soluble mediator so the oxidation of the mediator is limited to only one cycle per label for Ru(bpy)32+ and Os(bpy)32+. The method of Fowlkes and Thorp is distinctly different from the technology described in this application in that the electrochemical label is a transition metal complex in Fowlkes and Thorp, whereas the present invention provides a different label. When the electrochemical label is a transition metal complex, the number of cycles of mediated electron transfer is generally limited to one per label.
Measurement of a target protein was first achieved by Yalow and Berson (Yalow, R. S. and Berson, S. A., Nature, 1959, 184, 1648) using a competitive, radiolabeled ligand immunoassay (i.e., RIA) for the protein insulin. Since then numerous other labels have been employed in immunoassays such as enzymes, chemiluminescent and fluorescent labels, metal atoms, transition metal complexes and particles (i.e., polystyrene, gold) to measure selected target proteins (Tijssen, P., In: Laboratory Techniques in Biochemistry and Molecular Biology, 1985, Vol. 15. Elsevier Science Publishers, N.Y. 549 p). More recently, numerous other types of macromolecules other than immunoglobulins such as cell receptors (Guyda, H. J., J. Clin. Endocrinol. Metab., 1975, 41, 953; Strosberg, A. D., et al., Curr. Opin. Biotechnol., 1991, 2, 30), proteoglycans (Najjam, S., Cytokine, 1997, 9, 1013), extracellular matrix proteins (Mould, A. P., Meth. Mol Biol., 2000, 139, 295) and nucleic acids (McGown L. B., et al., Anal. Chem., 1995, 67, 663A) have been used as affinity binders in assays to detect target proteins. The instant invention to be described herein has the ability to utilize all the above molecular interactions to detect preselected target proteins as well as other binding substances by using peptide/amino acid or oligonucleotide labels in assays with catalytic transition metal mediated electrochemical detection.
Recently developed protein detection assay systems are commercially available from IGEN (Gaithersburg, Md.) and Luminex (Austin, Tex.). However, each of these systems is readily distinguishable from the present invention since they are dependent upon the teachings of electrochemiluminescence (IGEN) or fluorescent bead sorting (Luminex).
It is an object of this invention to detect binding interactions. The detection methodology involves oxidation of labels using transition metal mediator complexes in a detectable catalytic redox reaction and applies generally to binding interactions of immunoglobulins, receptors, proteins, and oligonucleotides with proteins, protein fragments, ligands, carbohydrates, drugs, drug candidates, steroids, hormones, and other substances. The labels are attached directly to the binders, target molecules, surrogate target molecules or to binders capable of binding targets or surrogate target molecules that compete with the target for binding. The labels can be naturally attached in the target, surrogate target, or binder (i.e., endogenous) or constructed by the covalent attachment of the label to the target, binder, or surrogate target (i.e., exogenous). Preferred labels include peptides and oligonucleotides. Preferred types of binding affinities to be utilized in the instant invention to detect target substances are based on antibodyxe2x80x94antigen, receptor (eukaryotic, prokaryotic or viral)xe2x80x94ligand, DNAxe2x80x94protein, drug targetxe2x80x94drug, and proteinxe2x80x94protein interactions.
It is a further object of this invention to use detected binding interactions between specific biomolecules to determine their presence (or absence) in test samples, such as clinical samples, environmental samples, pharmaceutical samples and others. It is an additional object of this invention to determine the impact of specific drugs on the detected binding interactions between biomolecules.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.
The invention herein provides a method of detecting binding interactions and target molecules, such as proteins, protein fragments, recombinant proteins, recombinant protein fragments, extracellular matrix proteins, ligands, carbohydrates, steroids, hormones, drugs, drug candidates, immunoglobulins and receptors of eukaryotic, prokaryotic or viral origin, by mediated electrochemistry using labels that react with transition metal mediator complexes in a detectable catalytic redox reaction. These labels are attached directly to binders, target molecules, surrogate target molecules, or to affinity ligands capable of binding to the target or to surrogate target molecules capable of competing with the target for binding to another binder. The labels can be naturally present (endogenous) in the binder, target or affinity ligand, or constructed by the covalent attachment of the label to the binder, target, affinity ligand or surrogate target (exogenous). The biological binding interactions detected with this technology include antibody-antigen, protein-protein, protein-nucleic acid, drug target-drug, and receptor-ligand interactions.
Other objects and features of the inventions will be more fully apparent from the following disclosure and appended claims.